Design, synthesis, and evaluation of some novel N-benzothiazol-2-yl benzamide derivatives as allosteric activators of human glucokinase

of this article was presented at CUDC consortium and Summer School Conference at Chitkara University, India. __________________ Arora et al. / Journal of Applied Pharmaceutical Science 11 (Supp 1); 2021: 038-047 039 allosteric location of GK (Grewal et al., 2014; Pal et al., 2009b). Some of the benzamide derivatives reported recently as potent GK activators are shown in Figure 1 along with their GK activity (Bowler et al., 2013; Charaya et al., 2018; Ericsson et al., 2012; Grewal et al., 2019a, 2019b; Lei et al., 2015; McKerrecher et al., 2018; Park et al., 2013, 2014; Pike et al., 2011; Sjostrand et al., 2013; Wang et al., 2017). Based on the above-mentioned facts, few newer N-benzothiazol-2-yl benzamide correspondents were proposed as potential activators of human GK. MATERIALS AND METHODS All the chemicals were acquired from reputed companies, including Spectrochem, Sisco Research Laboratories Pvt. Ltd. (SRL), S.D. Fine-Chem, Merck, Fisher Scientific, and SigmaAldrich etc., and employed without purification. Melting points of the synthesized molecules were determined using the uncorrected Veego Model of melting point apparatus melting point device. Culmination of reaction was checked employing silica Gel-G Thin layer chromatography (TLC). IR spectra were obtained using “Shimadzu Fourier-transform infrared (FTIR) spectrophotometer” (employing “KBr pellet” procedure). “Avance-II (Bruker) 400 MHz NMR spectrophotometer” was employed for taking Proton nuclear magnetic resonance (1H-NMR) and Carbon nuclear magnetic resonance (13C-NMR) spectra consuming appropriate dutereated solvent and conferred in parts per million (δ, ppm) downfield from tetramethylsilane (internal reference). General procedure for preparation of designed molecules Dry benzoic acid (1 mmol) was added to a flat bottom flask fixed on a magnetic stirrer at constant temperature around 10°C. Excess of sulfurochloridic acid (8.0 ml) was added carefully and observed to avoid any escape. When all the acid was liquefied and the exothermic response terminated, the flat bottom flask was heated at 70–80°C using water bath for 2 hours, followed by cooling. The materials of the flask were poured into crushed ice (150 g) with stirring and crystals of 3-(chlorosulfonyl)benzoic Figure 1. Some of the recently reported benzamide derivatives as potent GK activators. Arora et al. / Journal of Applied Pharmaceutical Science 11 (Supp 1); 2021: 038-047 040 acid were filtered employing vacuum subsequent to cold water wash, followed by air drying. The precipitates prepared earlier (1 mmol) were then reacted with the corresponding aliphatic and aromatic amines (1 mmol) under reflux using acetone till completion of the reaction (observed using TLC), following cooling and drying of the precipitates. The different sulfonamides prepared earlier (1 mmol) were refluxed with sulfinyl chloride (1 mmol) for 3 hours and extra sulfinyl chloride was removed to obtain the analogous acid chlorides. Acid chlorides prepared earlier (1 mmol) were refluxed with 2-aminobenzothiazole (1.5 mmol). The end products obtained by evaporation of solvent were purified using recrystallization from ethanol (Grewal et al., 2017; Singh et al., 2017). N-(1,3-benzothiazol-2-yl)-3-(phenylsulfamoyl)benzamide (1) FTIR (KBr Pellets) ν cm−1: 3,867.78 (NH str.), 3,737.50 (NH str.), 3,432.08.46 (NH str.), 2,973.38 (CH str.), 1,642.58 (C=O str.), 1,558.12 (NH bend), 1,463.36 (C=N str.), 1,417.54 (C=C str.), 1,296.70 (SO2 asym. str.), 1,076.13 (SO2 sym. str.), 752.08 (CH bend), 667.04 (C-S str.); 1H-NMR (δ ppm, 400 MHz, Dimethyl sulfoxide (DMSO-d6)): 8.92 (s, 1H, NH), 8.24–8.52 (m, 4H, CH), 7.34–8.26 (m, 4H, CH), 6.88–7.32 (m, 5H), 2.58 (s, 1H, NH); 13C-NMR (δ ppm, DMSO-d6): 172.09 (C=N), 164.46 (C=O), 152.34 (C), 136.32 (C), 135.38 (C), 131.78 (CH), 130.02 (C), 128.32 (C), 127.13 (CH), 125.23 (CH), 123.43 (CH), 122.02 (CH), 119.17 (CH), 118.64 (CH). N-(1,3-Benzothiazol-2-yl)-3-[(2-chloro-4-nitrophenyl) sulfamoyl]benzamide (2) FTIR (KBr Pellets) ν cm−1: 3,836.20 (NH str.), 3,446.91 (NH str.), 2,928.28 (CH str.), 1,641.34 (C=O str.), 1,632.23 (NH bend), 1,551.35 (C=N str.), 1,464.11 (NO2 sym. str.), 1,413.94 (NO2 asym. str.), 1,299.66 (SO2 asym. str.), 1,079.66 (SO2 sym. str.), 684.36 (C-Cl str.), 667.10 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 9.04 (s, 1H, NH), 8.02–8.48 (m, 4H, CH), 7.54–8.11 (m, 4H, CH), 6.98–8.12 (m, 3H, CH), 2.46 (s, 1H, NH); 13C-NMR (δ ppm, DMSO-d6): 169.78 (C=N), 162.32 (C=O), 151.56 (C), 139.22 (C), 137.28 (C), 133.67 (C), 131.09 (CH), 130.88 (C), 129.07 (CH), 128.57 (C), 127.46 (CH), 126.27 (C), 125.18 (CH), 124.22 (CH), 122.24 (CH), 118.88 (CH), 115.16 (CH). N-(1,3-benzothiazol-2-yl)-3-(benzylsulfamoyl)benzamide (3) FTIR (KBr Pellets) ν cm−1: 3,755.80 (NH str.), 3,448.08 (NH str.), 2,996.40 (CH str.), 2,912.85 (CH str.), 1,659.53 (C=O str.), 1,429.38 (NH bend), 1,311.51 (SO2 asym. str.), 1,025.25 (SO2 sym. str.), 696.02 (CH bend), 527.06 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 8.98 (s, 1H, NH), 8.16–8.42 (m, 4H, CH), 7.68–8.32 (m, 4H, CH), 7.16–7.58 (m, 5H, CH), 6.24 (t, 1H, NH), 4.42 (d, 1H, CH2); 13C-NMR (δ ppm, DMSO-d6): 176.02 (C=N), 165.32 (C=O), 152.34 (C), 141.46 (C), 140.06 (C), 134.45 (C), 130.34 (C), 130.12 (CH), 127.24 (CH), 124.37 (CH), 122.10 (CH), 119.43 (CH), 117.13 (CH), 48.32 (CH). N-(1,3-Benzothiazol-2-yl)-3-(butylsulfamoyl)benzamide (4) FTIR (KBr Pellets) ν cm−1: 3,754.38 (NH str.), 3,448.26 (NH str.), 2,930.77 (CH str.), 2,962.66 (CH str.), 1,643.35 (C=O str.), 1,554.13 (NH bend), 1,464.82 (C=C str.), 1,415.35 (SO2 asym. str.), 1,076.78 (SO2 sym. str.), 666.78 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 9.08 (s, 1H, NH), 7.80–8.12 (m, 4H, CH), 7.63–8.38 (m, 4H, CH), 5.54 (t, 1H, NH), 4.08 (m, 2H, CH2), 2.62 (m, 2H, CH2), 1.78 (m, 2H, CH2), 1.08 (m, 3H, CH3); 13C-NMR (δ ppm, DMSO-d6): 174.46 (C=N), 164.98 (C=O), 152.49 (C), 139.58 (C), 135.13 (C), 131.23 (CH), 130.34 (CH), 129.22 (CH), 125.47 (CH), 124.78 (CH), 121.12 (CH), 119.32 (CH), 118.34 (CH), 115.66 (CH), 45.08 (CH), 34.25 (CH), 20.88 (CH), 15.46 (CH). N-(1,3-Benzothiazol-2-yl)-3-(methylsulfamoyl)benzamide (5) FTIR (KBr Pellets) ν cm−1: 3,798.48 (NH str.), 3,448.44 (NH str.), 3,017.57 (CH str.), 2,966.14 (CH str.), 1,654.21 (C=O str.), 1,598.09 (NH bend), 1,544.68 (C=N str.), 1,388.45 (SO2 asym. str.), 1,189.77 (SO2 sym. str.), 789.65 (CH bend), 665.88 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 8.88 (s, 1H, NH), 7.94–8.43 (m, 4H, CH), 7.45–8.16 (m, 4H, CH), 5.34 (t, 1H, NH), 2.44 (s, 3H, CH3); 13C-NMR (δ ppm, DMSO-d6): 175.36 (C=N), 165.23 (C=O), 153.02 (C), 139.14 (C), 135.35 (C), 133.89 (CH), 133.13 (C), 132.06 (CH), 128.98 (CH), 125.83 (CH), 124.24 (CH), 121.56 (CH), 119.18 (CH), 118.11 (CH), 33.29 (CH). N-(1,3-benzothiazol-2-yl)-3-[(2-methylphenyl)sulfamoyl] benzamide (6) FTIR (KBr Pellets) ν cm−1: 3,791.96 (NH str.), 3,456.56 (NH str.), 3,013.67 (CH str.), 2,912.67 (CH str.), 1,667.25 (C=O str.), 1,604.66 (NH bend), 1,578.56 (C=N str.), 1,345.34 (SO2 asym. str.), 1,103.78 (SO2 sym. str.), 850.55 (CH bend), 664.89 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 8.89 (s, 1H, NH), 8.08–8.43 (m, 4H, CH), 7.42–8.08 (m, 4H, CH), 6.44–7.25 (m, 4H, CH), 2.54 (s, 1H, NH), 2.43 (s, 3H, CH3); 13C-NMR (δ ppm, DMSO-d6): 175.69 (C=N), 166.96 (C=O), 152.68 (C), 139.46 (C), 138.12 (C), 135.42 (C), 134.08 (C), 134.87 (CH), 133.67 (C), 132.12 (CH), 129.61 (CH), 126.59 (CH), 124.32 (CH), 123.56 (CH), 121.27 (CH), 118.25 (CH), 18.37 (CH). N-(1,3-benzothiazol-2-yl)-3-[(4-bromophenyl)sulfamoyl] benzamide (7) FTIR (KBr Pellets) ν cm−1: 3,837.14 (NH str.), 3,732.98 (NH str.), 3,441.64 (NH str.), 2,974.87 (CH str.), 1,641.67 (C=O str.), 1,553.91 (NH bend), 1,464.33 (C=N str.), 1,415.88 (SO2 asym. str.), 1,296.76 (SO2 sym. str.), 809.70 (CH bend), 753.82 (C-Br str.), 665.74 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 8.75 (s, 1H, NH), 8.14–8.38 (m, 4H, CH), 6.85–8.03 (m, 4H, CH), 7.06–7.38 (m, 4H, CH), 2.59 (s, 1H, NH); 13C-NMR (δ ppm, DMSO-d6): 176.08 (C=N), 166.34 (C=O), 153.12 (C), 139.10 (C), 137.05 (C), 134.28 (C), 132.72 (CH), 130.06 (CH), 129.73 (CH), 127.32 (CH), 124.94 (CH), 121.74 (CH), 120.21 (CH), 116.36 (C). N-(1,3-benzothiazol-2-yl)-3-[(4-nitrophenyl)sulfamoyl] benzamide (e8) FTIR (KBr Pellets) ν cm−1: 3,870.59 (NH str.), 3,755.40 (NH str.), 3,452.66 (NH str.), 2,997.49 (CH str.), 1,708.27 (C=O str.), 1,429.03 (NO2 sym. str.), 1,362.55 (NO2 asym. str.), 1,311.98 Arora et al. / Journal of Applied Pharmaceutical Science 11 (Supp 1); 2021: 038-047 041 (SO2 asym. str.), 1,223.02 (SO2 sym. str.), 696.02 (CH bend), 526.51 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 8.84 (s, 1H, NH), 8.28–8.58 (m, 4H, CH), 7.89–8.17 (m, 4H, CH), 6.68– 7.85 (m, 4H, CH), 2.50 (s, 1H, NH); 13C-NMR (δ ppm, DMSO-d6): 174.24 (C=N), 165.89 (C=O), 153.12 (C), 143.04 (C), 139.16 (C), 137.32 (C), 134.04 (C), 133.89 (C), 129.82 (CH), 124.22 (CH), 121.01 (CH), 120.84 (CH), 119.25 (CH), 116.39 (CH). N-(1,3-benzothiazol-2-yl)-3-[(4-methylphenyl)sulfamoyl] benzamide (9) FTIR (KBr Pellets) ν cm−1: 3,868.16 (NH str.), 3,754.28 (NH str.), 3,448.36 (NH str.), 2,930.77 (CH str.), 1,643.31 (C=O str.), 1,553.03 (NH bend), 1,464.83 (C=N str.), 1,415.35 (CH bend), 1,300.62 (SO2 asym. str.), 1,076.79 (SO2 sym. str.), 717.52 (CH bend), 666.77 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 8.76 (s, 1H, NH), 7.98–8.28 (m, 4H, CH), 7.32–7.75 (m, 4H, CH), 6.32–7.23 (m, 4H, CH), 2.51 (s, 1H, NH), 2.36 (s, 3H, CH3); 13C-NMR (δ ppm, DMSO-d6): 176.03 (C=N), 165.94 (C=O), 153.12 (C), 139.88 (C), 136.65 (C), 135.14 (C), 133.74 (C), 132.64 (C), 130.95 (CH), 129.18 (CH), 125.34 (CH), 124.04 (CH), 120.84 (CH), 119.23 (CH), 118.06 (CH), 25.98 (CH). N-(1,3-Benzothiazol-2-yl)-3-(propylsulfamoyl)benzamide (10) FTIR (KBr Pellets) ν cm−1: 3,450.06 (NH str.), 2,996.68 (NH str.), 2,912.98 (CH str.), 1,689.51 (C=O str.), 1,428.92 (NH bend), 1,311.73 (C=N str.), 1,023.65 (SO2 asym. str.), 950.47 (SO2 sym. str.), 696.27 (CH bend), 524.89 (C-S str.); 1H-NMR (δ ppm, 400 MHz, DMSO-d6): 9.11 (s, 1H, NH), 8.20–8.43 (s, 3H, CH), 7.58–8.12 (m, 4H, CH), 6.34 (t, 1H, NH), 3.94 (m, 2H, CH2), 2.65 (m, 2H, CH2), 1.24 (t, 3H, CH3); 13C-NMR (δ ppm, DMSO-d6): 175.24 (C=N), 165.14 (C=O), 154.23 (C), 138.42 (C), 134.64 (C), 130.54 (C), 129.18 (CH), 125.26 (CH), 120.47 (CH), 119.08 (CH), 117.56 (CH), 44.28 (CH), 31.08 (CH), 16.22 (CH).


INTRODUCTION
Type 2 diabetes (T2D) is a life-long food metabolism ailment owing to decreased insulin action resulting in hyperglycemia and is prevalent among most of the patients suffering from diabetes (Bastaki, 2005;Kohei, 2010;Olokoba et al., 2012). Although ample types of oral antidiabetic agents are available to be used in the management of T2D, no individual antidiabetic agent is valuable in attaining persistent homeostasis of plasma sugar within usual physiological range in majority of the persons suffering from T2D. Owing to the above-mentioned points, nowadays doctors advise a combination of hypoglycemic agents in the initial phase of T2D treatment. Additionally, overdose of hypoglycemic drugs may possibly result in serious hypoglycemia triggering brutal adverse reactions, and patients generally require urgent medical treatment (Grewal et al., 2016b;Olokoba et al., 2012;Pal, 2009b). Nowadays, medicinal chemistry scientists are aiming at designing newer effective hypoglycemic agents having distinct mechanisms of action at the molecular level which could be used as a single drug with improved safety (Grewal et al., 2014(Grewal et al., , 2016a. Glucokinase (GK) is a cytoplasmic enzyme that is expressed predominantly in pancreatic β-cells and liver hepatocytes, and fastens the conversion of glucose to glucose-6phosphate with the help of adenosinetriphosphate (ATP) (Coghlan and Leighton, 2008;Pal, 2009a). In beta-cells of pancreas gland, GK regulates glucose-instigated discharge of insulin, and in liver hepatocytes of the liver, it commands the breakdown of sugars. GK acts as an emergent medication focus for treatment and management of T2D due to its key function in controlling sugar breakdown. Small molecule activators of human GK are the unique class of therapeutically useful agents that allosterically activate GK and illustrate their plasma sugar-lowering potential (Coghlan and Leighton, 2008;Grewal et al., 2014;Matschinsky et al., 2011;Perseghin, 2010). Several GK activators have been progressed into clinical trials (phase II), including AZD6370, AZD1656, MK-0941, Piragliatin, and AMG151; even though a strong decrease in blood sugar was observed, potential adverse reactions were reported, such as hypoglycemia and elevated levels of triglycerides. The literature survey revealed that most of the drug discovery and development research associated with allosteric activators of human GK were mainly focused on the substituted benzamide analogs probably owing to their corresponding alignment outline and bonding connections with the residues of allosteric location of GK (Grewal et al., 2014;Pal et al., 2009b). Some of the benzamide derivatives reported recently as potent GK activators are shown in Figure 1 along with their GK activity (Bowler et al., 2013;Charaya et al., 2018;Ericsson et al., 2012;Grewal et al., 2019aGrewal et al., , 2019bLei et al., 2015;McKerrecher et al., 2018;Park et al., 2013Park et al., , 2014Pike et al., 2011;Sjostrand et al., 2013;Wang et al., 2017). Based on the above-mentioned facts, few newer N-benzothiazol-2-yl benzamide correspondents were proposed as potential activators of human GK.

MATERIALS AND METHODS
All the chemicals were acquired from reputed companies, including Spectrochem, Sisco Research Laboratories Pvt. Ltd. (SRL), S.D. Fine-Chem, Merck, Fisher Scientific, and Sigma-Aldrich etc., and employed without purification. Melting points of the synthesized molecules were determined using the uncorrected Veego Model of melting point apparatus melting point device. Culmination of reaction was checked employing silica Gel-G Thin layer chromatography (TLC). IR spectra were obtained using "Shimadzu Fourier-transform infrared (FTIR) spectrophotometer" (employing "KBr pellet" procedure). "Avance-II (Bruker) 400 MHz NMR spectrophotometer" was employed for taking Proton nuclear magnetic resonance ( 1 H-NMR) and Carbon nuclear magnetic resonance ( 13 C-NMR) spectra consuming appropriate dutereated solvent and conferred in parts per million (δ, ppm) downfield from tetramethylsilane (internal reference).

General procedure for preparation of designed molecules
Dry benzoic acid (1 mmol) was added to a flat bottom flask fixed on a magnetic stirrer at constant temperature around 10°C. Excess of sulfurochloridic acid (8.0 ml) was added carefully and observed to avoid any escape. When all the acid was liquefied and the exothermic response terminated, the flat bottom flask was heated at 70-80°C using water bath for 2 hours, followed by cooling. The materials of the flask were poured into crushed ice (150 g) with stirring and crystals of 3-(chlorosulfonyl)benzoic acid were filtered employing vacuum subsequent to cold water wash, followed by air drying. The precipitates prepared earlier (1 mmol) were then reacted with the corresponding aliphatic and aromatic amines (1 mmol) under reflux using acetone till completion of the reaction (observed using TLC), following cooling and drying of the precipitates. The different sulfonamides prepared earlier (1 mmol) were refluxed with sulfinyl chloride (1 mmol) for 3 hours and extra sulfinyl chloride was removed to obtain the analogous acid chlorides. Acid chlorides prepared earlier (1 mmol) were refluxed with 2-aminobenzothiazole (1.5 mmol). The end products obtained by evaporation of solvent were purified using recrystallization from ethanol Singh et al., 2017).

In vitro GK assay
GK activation potential of all the derivatives was assessed by employing a combined response with glucose-6phosphate dehydrogenase (G6PDH) using spectrometry (Efanov et al., 2005;Futamura et al., 2006;Grewal et al., 2019c). All the samples were made using DMSO and the in-vitro GK test was conducted in a final volume of 2,000 µl comprising 0.25 mM 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 10 mM dextrose, 25 mM KCl, 1 mM MgCl 2 , 1 mM 1,4-dithio-D-threitol, 1 mM nicotinamide adenine dinucleotide, 1 mM ATP, G6PDH (2.5 U/ml), 0.5 µg GK, and derivatives to be tested (10 µM). Readings were taken at 340 nm following a nurture time of 3 minutes and GK activation was computed in comparison to DMSO (activation of GK by DMSO alone was treated as 100%). All the results were represented as mean (n = 3) ± standard deviation. The in vitro GK assay data for test groups were statistically analyzed by one-way analysis of variance for comparison and significance from control group (value of p < 0.05) using GraphPad Prism (GraphPad Software Inc.).

In silico prediction of pharmacokinetic parameters
All the designed molecules were analyzed for the prediction of pharmacokinetic parameters related to absorption, distribution, metabolism, and excretion (ADME) by employing FAF-Drugs4 server; and accessed using Lipinski's rule of 5 for drug-likeness (Lagorce et al., 2017;Miteva et al., 2006).

Molecular docking investigations
Molecular docking investigations were carried out for all the analogs in the allosteric binding location of the GK employing AutoDock Vina and AutoDock Tools (ADTs) (Morris et al., 2009;Trott and Olson, 2010). The 2D chemical structures of all the ligands were prepared by MarvinSketch 18.5.0 (ChemAxon), followed by conversion to 3D by Frog2 (Miteva et al., 2010). The ligands were converted into "pdbqt" files using ADTs. After assessing a number of co-crystallized structures for GK available in the protein data bank; the best ligand bound complex was selected based on higher resolution and key binding interactions between the GK and small molecule activators (Protein data bank (PDB) ID: 3IMX). The PDB file of the GK protein were edited using PyMOL (Schrödinger, Limited liability company.) by removing the cocrystallized activator, entirely water molecules along with other non-interacting species. The "pdbqt" files of target proteins were produced from the PDB files using ADTs. The "grid" tool of ADTs was used to calculate the grid parameters and all the information concerning target protein, ligand, grid size, and geometry were saved in "txt" file. The reference ligand was docked with GK and compared with that of reference GK activator for determining accuracy of docking protocol. The 3D optimized ligands were docked with the refined GK protein and scored by scoring function. The binding free energy (ΔG, kcal/mol) for each compound was reported in a log file and the binding interactions of the ligands in binding site of the target proteins were analyzed using Name of graphics tool (Grewal et al., 2019b;Rathee et al., 2019).

In silico prediction of toxicity
All the compounds were evaluated in silico for the prediction of possible toxicity of these compounds using Name of tool used for prediction of pharmacokinetic properties online computer program (Pires et al., 2015(Pires et al., , 2018Salgueiro et al., 2016). Scheme 1. General synthetic route followed for N-benzothiazol-2-yl benzamide analogs. Reagents and conditions: (a) HClSO 3 ; (b) NH 2 -R 1 , reflux; (c) SO 2 Cl, reflux; (d) NH 2 -R 2 , reflux.

RESULTS AND DISCUSSION
Chemistry 3-(Chlorosulfonyl)benzoic acid attained via chlorosulfonation of benzoic acid was reacted with various amines to get the sulfonamides. These sulfonamides were then reacted with thionyl chloride, followed by reaction with 2-aminobenzothiazole to synthesize the desired compounds (Scheme 1) in good yield ( Table 1).
The 1 H-NMR spectra of the prepared analogues exhibited the singlet signal corresponding to 1 proton of CONH scaffold in the range δ 9-10 ppm, therefore, supporting the development of benzamide connection in these derivatives. The occurrence of singlet signal for 1 NH proton of sulfonamide moiety at about δ 2.5 ppm depicted the synthesis of sulfonamide analogues by the reaction of sulfonyl chloride derivatives with the corresponding amines. The existence of one singlet signal (equivalent to C 2 ), two doublet signals (corresponding to C 4 and C 6 ), and a triplet signal (corresponding to C 5 ) around δ 7.5-8 ppm of the benzamide scaffold established the fact that the benzamide and sulfonamide functional moieties were located meta to each other. Two doublets and two triplet signals corresponding to four protons (aromatic CH) were detected at about δ 7-8 ppm in the 1 H-NMR spectra of these molecules approving that the designed derivatives were synthesized by reacting benzoyl chloride with 2-aminobenzothiazole. In the 13 C-NMR spectra of the prepared molecules signals around δ 175 ppm indicated the existence of C=N bond in these derivatives and signals around δ 165 ppm indicated presence of amide C=O bond therefore supporting the formation of benzamide linkage in these derivatives. The FTIR spectra of these molecules exhibited NH-stretching vibrations (for amide) >3,500 cm −1 ; CH-stretching (aromatic) vibrations > 3,000 cm −1 ; SO 2 stretching vibrations (asymmetric and symmetric) in the range of 1,399-1,301 cm −1 and 1,199-1,101 cm −1 , respectively; and SO 2 NH stretching peaks at around 3,399-3,101 cm −1 , thus confirming the presence of a benzamide functional group (CONH) and a sulfonamide moiety these newly prepared analogs. In the FTIR spectra of these compounds stretching vibrations for C=O at around 1,699-1,601 cm −1 supported the occurrence of benzamide carbonyl moiety in the construction of these analogues. The occurrence of the NHbending vibrations near to 1,600 cm −1 depicted the presence of aromatic NH-moiety in the construction of these molecules.

In vitro GK assay
The outcomes of the in vitro GK test (activation of GK enzyme by the synthesized derivatives compared to control) are shown in Figure 2. Among the synthesized compounds evaluated, 1, 2, 6, and 7 revealed maximum GK activity in the GK test (GK activation fold in the range of 1.6-2.0 compared to control). Compounds 5 and 8 disclosed moderate GK activation (fold activation in the range of 1.3-1.5) of GK enzyme. Compounds 3 and 9 demonstrated poor GK activity (fold activation about 1.20) in comparison to that of control. Compounds 4 and 10 were unsuccessful in the in vitro GK assay.
Among the synthesized analogs, compounds bearing N-(2-methylphenyl) sulfonamide moiety (compound 6) exhibited highest GK activity (GK activation fold of 1.97). Derivative bearing N-4-bromophenyl substituted sulfonamide moiety (compound 7) displayed potent 1.84-fold activation in comparison to that of the control. Analogs having N-phenyl and N-2-chloro-4-nitrophenyl sulfonamide moiety (1 and 2, respectively) demonstrated 1.66 and 1.69-fold GK activation, correspondingly. Synthesized compounds having N-4-nitrophenyl and N-methyl sulfonamide moiety (5 and 8, respectively) exhibited moderate GK activity (1.34 and 1.44-fold GK activation, respectively). Analogs bearing N-benzyl and N-4methylphenyl sulfonamide moiety (3 and 9, respectively) exhibited poor GK activity (1.26 and 1.24-fold activation, correspondingly). Derivatives having N-butyl and N-propyl sulfonamide moiety (compounds 4 and 10, respectively) were ineffective in the GK assay. Outcomes of the GK test demonstrated that replacement of substituted phenyl moiety substituted to SO 2 NH resulted in improved GK activity compared to those having alkyl group as can be seen from GK activity of compounds 1, 2, 6, and 7. Substitution of aromatic moiety attached to SO 2 NH with alkyl chains led to reduced GK activation potential compared to compounds having aromatic moiety as can be seen from GK activity of compounds 4, 5, and 10.
Prediction of ADME properties ADME parameters, including molecular weight (MW), partition coefficient (log P), distribution coefficient (log D), water solubility (log S w ), "topological polar surface area" (tPSA), "H-bond acceptors" (HBA), "H-bond donors" (HBD), solubility (mg/l) and "number of rotatable bonds" (NRB), were predicted for all the designed compounds. All of the designed compounds showed good pharmacokinetic parameters for oral bioavailability (Table 2) and drug-like properties as described using "Lipinski's rule of 5" (Pfizer's rule of five or simply rule of five).

In silico docking investigations
The reference ligand of PDB ID of the protein structure used in docking studies was docked with GK; and the docked reference  Figure 2. GK activity (GK fold activation) of the synthesized derivatives (at 10-µM concentration). * Data were significantly dissimilar compared to control (p < 0.05). $ Data were not significantly dissimilar compared to control (p > 0.05).  GK activator produced an analogous bonding outline and overlay on the binding mode of the co-crystallized activator with ΔG of −9.0 kcal/mol validating accuracy of docking methodology. Most of the docked ligands exhibited considerable binding with allosteric site residues of GK as established by analyzing their bonding interactions and ΔG of the best docked poses. Based on their bottommost "ΔG" values and docking connections with the allosteric site residues; compounds 1, 2, 5, 6, 7, and 8 were additionally examined in minutiae using PyMOL for exploring binding interactions of these molecules with allosteric site residues of GK (Table 3). Super-positioning of the docked poses of 1, 2, 5, 6, 7, and 8 on that of reference ligand in the allosteric site of GK protein demonstrated that the these molecules had the analogous binding and orientation arrangement in the allosteric site of GK as that of the co-crystallized activator (PDB entry: 3IMX) ["(2R)-3-cyclopentyl-N-(5-methoxy[1,3]thiazolo[5,4-b]pyridin-2-yl]-2-{4-[(4methylpiperazin-1-yl)sulfonyl]phenyl}propanamide") supporting the outcomes of in vitro GK test for these compounds (Fig. 3).
The docked pose of compounds 1, 2, 5, 6, 7, and 8 showed H-bond interactions between "N" of benzothiazol-2-yl group and amide NH of Arg63 residues; and "benzamide NH" group and "backbone C=O" of Arg63 residue in the allosteric location of GK with bond length in the range 3.0 Å-4.1 Å; and 3.0 Å-3.3 Å, respectively. Overall, the benzothiazol-2-yl moiety bonded to the benzamide "NH" of these compounds projected in the hydrophobic cavity displaying connections with the Val455 and Lys459 amino acid residues, along with Pro66 and Ile159 amino acid residues, aromatic moiety parceled in the cavity composed of Met210, Tyr214, and Val455 residues (Fig. 4).

In silico prediction of toxicity
The possible toxicity (mutagenic, cardiotoxicity, acute toxicity, hepatotoxicity, skin irritation, and chronic toxicity) for the optimized compounds was accessed using the pkCSM online platform. Conferring to the results represented in Table 4, all the compounds showed little toxicity probability. For all the compounds accessed in silico for prediction of toxicity using the online program, mutagenicity was predicted for compounds 2 and 8. In this perspective, the initial evaluation performed in silico can supplement forthcoming studies related to the safety of these compounds.

CONCLUSION
In summary, a series of novel N-benzothiazol-2-yl benzamide derivatives were designed and synthesized based on structure-based drug design approach. Among these newly identified derivatives, analogs 6 and 7 unveiled maximum GK activation potential (>1.6-fold GK activation). Outcomes of the in-vitro GK test were found to be analogous to the in-silico docking investigations with the GK enzyme. These freshly developed compounds can assist in finding the lead analogues for ultimate discovery of strong and harmless activators of GK for T2D handling and management.